Actinomycete integrative and conjugative elements

This paper reviews current knowledge on actinomycete integrative and conjugative elements (AICEs). The best characterised AICEs, pSAM2 of Streptomyces ambofaciens (10.9 kb), SLP1 (17.3 kb) of Streptomyces coelicolor and pMEA300 of Amycolatopsis methanolica (13.3 kb), are present as integrative elements in specific tRNA genes, and are capable of conjugative transfer. These AICEs have a highly conserved structural organisation, with functional modules for excision/integration, replication, conjugative transfer, and regulation. Recently, it has been shown that pMEA300 and the related elements pMEA100 of Amycolatopsis mediterranei and pSE211 of Saccharopolyspora erythraea form a novel group of AICEs, the pMEA-elements, based on the unique characteristics of their replication initiator protein RepAM. Evaluation of a large collection of Amycolatopsis isolates has allowed identification of multiple pMEA-like elements. Our data show that, as AICEs, they mainly coevolved with their natural host in an integrated form, rather than being dispersed via horizontal gene transfer. The pMEA-like elements could be separated into two distinct populations from different geographical origins. One group was most closely related to pMEA300 and was found in isolates from Australia and Asia and pMEA100-related sequences were present in European isolates. Genome sequence data have enormously contributed to the recent insight that AICEs are present in many actinomycete genera. The sequence data also provide more insight into their evolutionary relationships, revealing their modular composition and their likely combined descent from bacterial plasmids and bacteriophages. Evidence is accumulating that AICEs act as modulators of host genome diversity and are also involved in the acquisition of secondary metabolite clusters and foreign DNA via horizontal gene transfer. Although still speculative, these AICEs may play a role in the spread of antibiotic resistance factors into pathogenic bacteria. The novel insights on AICE characteristics presented in this review may be used for the effective construction of new vectors that allows us to engineer and optimise strains for the production of commercially and medically interesting secondary metabolites, and bioactive proteins

IDENTIFICATION AND FUNCTIONAL-ANALYSIS OF THE TRANSFER REGION OF PLASMID PMEA300 OF THE METHYLOTROPHIC ACTINOMYCETE AMYCOLATOPSIS-METHANOLICA

Amycolatopsis methanolica contains a 13.3-kb plasmid (pMEA300) that is present either as an integrated element or as an autonomously replicating plasmid. Conjugational transfer of pMEA300 results in pock formation, zones of growth inhibition that become apparent when plasmid-carrying donor cells develop in a confluent lawn of plasmid-lacking recipient cells. A 6,2-kb pMEA300 DNA region specifying the functions of conjugation and pock formation was sequenced, revealing 10 open reading frames, This is the first sequence of the transfer region of a plasmid from a nonstreptomycete actinomycete. No clear similarities were found between the deduced sequences of the 10 putative Tra proteins of pMEA300 and those of Streptomyces plasmids, All Tra proteins of pMEA300 thus may represent unfamiliar types, A detailed mutational analysis showed that at least four individual proteins, TraG (9,488 Da), TraH (12,586 Da), TraI (40,468 Da), and TraJ (81,109 Da), are required for efficient transfer of pMEA300, Their disruption resulted in a clear reduction in the conjugational transfer frequencies, ranging from (5.2 x 10(1))-fold (TraG) to (2.3 x 10(6))-fold (TraJ), and in reduced pock sizes, At least two putative proteins, TraA (10,698 Da) and TraB (31,442 Da), were shown to be responsible for pock formation specifically, Specific binding of the pMEA300-encoded KorA protein to the traA-korA intragenic region was observed

A plasmid from the methylotrophic actinomycete amycolatopsis-methanolica capable of site-specific integration

Amycolatopsis methanolica contains a 13.3-kb plasmid (pMEA300) which is present both in the free state and integrated at a unique genomic location. A 2.1-kb pMEA300 DNA fragment was sequenced, revealing the putative attP site and two open reading frames, xis and int, showing similarity to genes encoding excisionases and integrases, respectively